Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

Abstract

Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistancegene product is available.adaptive evolution ͉ molecular recognition ͉ plant-pathogen interactions ͉ protein-protein interactions ͉ polygalacturonase-inhibiting protein (PGIP)

Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering

“…Remarkably, many residues that were previously selected and validated as hot spots for this interaction (Casasoli et al, 2009) were found at the protein-protein interface of the complex (see "Discussion"). The active site of FpPG is located in a deep cleft that is open on both sides to allow the accommodation of the substrate (homogalacturonan) and the release of the hydrolysis products.…”

“…Genes encoding PGIPs are under selective pressure for diversification and a number of hot spots for the interaction with PGs have been identified on the inhibitor concave surface and validated by site-directed mutagenesis (Casasoli et al, 2009). Unlike for other CWDE inhibitors that engage many contacts in the interaction with their partners (Payan et al, 2004;Raiola et al, 2004;Di Matteo et al, 2005), only a few residues, sometimes only one, are critical for maintaining the stability of the PG-PGIP interaction (Leckie et al, 1999;Casasoli et al, 2009). This is probably why, despite the efforts made during the past years, the crystal structure of the complex is not available yet.…”

A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides

“…This is due to the bridging by Ca ++ of antiparallel HGA chains with continuous runs of dissociated and negatively charged carboxyl groups to form structures called "egg-boxes" (4-6). Fragments of HGA, named oligogalacturonides (OGs), function as danger signals and induce the expression of defense genes and proteins (5,(7)(8)(9)(10), protecting plants against fungal diseases (11). Besides inducing defense responses, OGs also affect several aspects of plant growth and development (5,12).…”

Arabidopsis MPK3 and MPK6 Play Different Roles in Basal and Oligogalacturonide- or Flagellin-Induced Resistance against Botrytis cinerea

“…Well-characterized DAMPs are the oligogalacturonides (OGs), pectin fragments released from the plant cell wall by fungal polygalacturonases (Hahn et al, 1981;Ridley et al, 2001). The accumulation of active OGs, with a degree of polymerization between 10 and 15, is favored by the presence of polygalacturonase-inhibiting proteins in the apoplast (De Lorenzo and Ferrari, 2002;Casasoli et al, 2009). OGs elicit a variety of responses, including the production of reactive oxygen species (ROS; Bellincampi et al, 2000;Galletti et al, 2008), a rapid modification of membrane polarization and ion fluxes (Mathieu et al, 1991;Thain et al, 1995), the induction of defense-related genes, and the accumulation of phytoalexins (Davis et al, 1986).…”